Modified oligonucleotides comprising thiol functions and use thereof for detecting nucleic acids

Patent metadata

Field	Value
Publication number	US-11390643-B2
Application number	US-201916271581-A
Country	US
Kind code	B2
Filing date	Feb 8, 2019
Priority date	Apr 4, 2012
Publication date	Jul 19, 2022
Grant date	Jul 19, 2022

Abstract

The present invention relates to a modified oligonucleotide having two or more thiol functions, which can be immobilized on a gold surface or on a grafted surface, in particular a surface comprising at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, preferably maleimide or acrylamide functions. The invention also relates to a method for detecting a nucleic acid in a biological sample comprising a step of detecting hybridization between a modified oligonucleotide and a target nucleic acid amplified from the biological sample. The invention relates more particularly to a method for detecting, genotyping or sequencing a pathogenic organism, preferably a virus.

First claim

What is claimed is:

1. A modified oligonucleotide corresponding to the formula:

in which,
n is an integer ranging from 4 to 100,
y is an integer ranging from 2 to 12,
N 1 , . . . , N n represent, independently of one another, a nucleotide,
X is selected from the group consisting of linear or branched C1-C12 alkyl groups, C1-C12 aminoalkyl groups, C1-C12 alkoxy groups, C3-C12 cycloalkyl groups, and oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups,
Y is selected from the group consisting of linear or branched C1-C12 alkyl groups, C1-C12 aminoalkyl groups, C1-C12 alkoxy groups, C3-C12 cycloalkyl groups, and oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups,
Z is selected from the group consisting of C1-C12 alkoxy groups, oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups, C1-C12 NCO-alkyl groups, and C1-C12 CON-alkyl groups,
W is selected from the group consisting of C1-C12 alkane triyl groups, C6-C18 aryl triyl groups, and C6-C18 aralkane triyl groups,
R is H or is selected from the group consisting of C1-C12 acyl, C1-C12 S-alkyl, C6-C12 S-aryl, S-2-pyridine, oxygen-containing or nitrogen-containing C1-C12 S-heteroalkyl, C3-C12 S-cycloalkyl, and oxygen-containing or nitrogen-containing C3-C12 S-cycloheteroalkyl groups, and
B n represents the base of the n-th nucleotide.

2. The modified oligonucleotide according to claim 1 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) is specific to a virus, a bacterium or a gene responsible for or involved in a disease.

3. The modified oligonucleotide according to claim 2 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) is selected from the group consisting of:
the sequences SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 35 and SEQ ID NO: 36 specific to the hepatitis C virus (HCV),
the sequences SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 40, specific to the flaviviruses,
the sequence SEQ ID NO: 18 and SEQ ID NO: 41, specific to the dengue viruses, and
the sequence SEQ ID NO: 19, specific to the West Nile viruses (WNV).

4. The modified oligonucleotide according to claim 1 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) has a structure of the alpha anomer, beta anomer, linear, or “snail” type.

5. A grafted substrate comprising at least one modified oligonucleotide according to claim 1 , said substrate comprising at least one receiving zone coated with a substance that tolerates the grafting of said modified oligonucleotide.

6. The grafted substrate according to claim 5 , in which:
said receiving zone is coated with a gold or platinum film, and said substrate is of metal, or
said receiving zone comprises on its surface at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, and said substrate is of plastic.

7. The grafted substrate according to claim 5 , in which said substrate is non-planar.

8. A method for detecting at least one target nucleic acid, comprising a step of:
detecting said at least one nucleic acid with the grafted substrate according to claim 5 .

9. A method for detecting at least one target nucleic acid in a biological sample, comprising a step of:
detecting said target nucleic acid with at least one detection probe formed by a modified oligonucleotide according to claim 1 .

10. The method according to claim 9 , comprising the steps of:
obtaining at least one source nucleic acid from said biological sample,
producing an amplicon by the amplification of said target nucleic acid from the source nucleic acid, and
detecting the hybridization of said amplicon with at least one detection probe formed by a modified oligonucleotide according to claim 1 .

11. The method according to claim 10 , for determining a genotype and/or subtype of a virus present in a biological sample, in which
the amplicon is generated by the amplification of a target nucleotide sequence, corresponding to a genomic region of virus bearing information relating to the genotype and/or subtype, and
detection of the hybridization of said amplicon with said at least one detection probe is carried out with a probe specific to a viral genotype and/or subtype.

12. The method according to claim 11 , in which the step of production of the amplicon is carried out by amplifying a target nucleotide sequence corresponding to a genomic region of the virus bearing information relating to the viral genotype and/or subtype, with a mixture of nucleotide primer pairs selected from the group consisting of:
SEQ ID NO: 8 and SEQ ID NO: 9, when the amplicon is generated starting from any genotype of HCV;
SEQ ID NO: 10 and SEQ ID NO: 9, when the amplicon is generated starting from an HCV of genotype 1a/1b;
SEQ ID NO: 29 and SEQ ID NO: 9, when the amplicon is generated starting from an HCV of genotype 2;
SEQ ID NO: 8 and SEQ ID NO: 11, when the amplicon is generated starting from an HCV of genotype 3a;
SEQ ID NO: 8 and SEQ ID NO: 30, when the amplicon is generated starting from an HCV of genotype 4a/4b; and
SEQ ID NO: 20 and SEQ ID NO: 21, or SEQ ID NO: 22 and SEQ ID NO: 21 when the amplicon is generated from a flavivirus.

13. The method according to claim 9 , wherein the method is applied for diagnostics, genotyping or sequencing of viral strains.

14. The method according to claim 13 , wherein the method is applied for diagnostics, genotyping or sequencing of HCV, HBV, dengue viruses or West Nile virus.

15. A kit for detecting at least one target nucleic acid in a biological sample comprising:
at least one modified oligonucleotide according to claim 1 and at least one substrate comprising at least one receiving zone coated with a substance that tolerates the grafting of said modified oligonucleotide, said receiving zone being coated with gold, with platinum or comprising on its surface at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, or
at least one grafted substrate according to claim 5 .

Assignees

• Francais Du Sang Ets
• Centre Nat Rech Scient
• Univ Montpellier
• Univ Claude Bernard Lyon

Inventors

• Fournier-Wirth Chantal
• Lereau Myriam
• Cantaloube Jean-François
• Vasseur Jean-Jacques
• Morvan François
• Meyer Albert
• Mayen Julie
• Chaix Carole
• Farre Carole

Classifications

• C12Q2600/158

  Expression markers · CPC title

• C07H21/00 Primary

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/70 Primary

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title

• C12Q1/707 Primary

  non-A, non-B Hepatitis, excluding hepatitis D · CPC title